1. Field of the Invention
The present invention relates to two DNA replication factors capable of enhancing DNA polymerase activity from Pyrococcus, and to a DNA polymerase reaction system utilizing such factors.
2. Background Art
DNA polymerase is an enzyme useful for DNA sequencing reaction, polymerase chain reaction (PCR), radioactive labeling of DNA, in vitro synthesis of a mutated gene, and the like. DNA polymerases currently known can be generally classified into six families based on their amino acid sequence homology. Among them, DNA polymerases usually used as reagents in gene manipulation experiments belong to Family A polymerases such as typical E. coli DNA pol I and thermophilic bacterium Thermus aquaticus DNA polymerase (i.e., Taq DNA polymerase), and Family B polymerases such as typical T4 phage DNA polymerase. Various DNA polymerases having different optimum temperatures have been discovered from bacteria as well as from animals and plants. However, many of them, derived from mesophilic organisms and thus having low thermostability, are not suitable, for example, for PCR comprising heat denaturation of template DNA at 94° C. or over.
Enzymes from thermophilic bacteria, such as Taq DNA polymerase, are commercially available as thermostable DNA polymerases. However, all of them lack 3′-5′ proofreading exonuclease activity, resulting in higher error rates during polymerase reactions such as PCR, and hence are not suitable for PCR with high fidelity and the like. Further, type B enzymes that are thermostable and have 3′-5′ proofreading exonuclease activity, are isolated from hyperthermophilic archaea such as Pyrococcus and Thermococcus, and are commercially available. However, they have low primer extension activity and so are not suitable for PCR for long-strand DNA.
Examples of PCR techniques developed so far include conventional PCR using commercially available thermostable Pol A or Pol B, and isothermal PCR using φ29 DNA polymerase having high strand-displacement activity. The region replicable by the conventional Pol A or Pol B enzymes is short, the maximum being about 10 kb. In addition, the synthesis rate is as low as 30 b/sec. On the other hand, when φ29 DNA polymerase is used, PCR can be conducted at ambient temperature, so no expensive apparatus for amplification reaction is required and the procedure is simple. However, the use of random primers causes replicated regions to be relatively short, so that it is difficult to synthesize or produce a long DNA strand. Further, it is difficult to amplify DNA directly from the blood, body fluid, etc., by using the above conventional enzymes. This is because any of those conventional enzymes exhibits a reduced activity of DNA synthesis at a high salt concentration, and hence desalting is required to lower the salt concentration in the reaction solution.
The present inventors, for the first time, discovered a DNA polymerase from Pyrococcus horikoshii, which is thermostable and has 3′-5′ proofreading exonuclease activity, and the gene thereof (JP Patent No. 3015878). Moreover, we successfully improved the DNA polymerase activity dramatically by removing an intein sequence from a large subunit of the DNA polymerase (JP Patent Publication (Kokai) No. 2001-299348A). Further, this DNA polymerase has a unique property that its primer extension activity becomes higher as the primer length is longer. However, this DNA polymerase also exhibits a reduced activity at a high salt concentration, although its activity is high at a low salt concentration.
Under these circumstances, the object of the present invention is to construct a new DNA polymerase reaction system utilizing a DNA polymerase, which is highly thermostable, has 3′-5′ exonuclease activity by which mistakes occurring in a newly extended DNA strand are corrected, and also exhibits a high primer extension activity, wherein the high DNA polymerase activity is exerted even at a high salt concentration. Thereby, a novel technology is provided wherein a long-strand DNA region having a length of several Mb can be quickly replicated even under a high salt concentration without pretreatment.